Improved efficiency M13 cloning using electroporation.

We compared the efficiency of transformation of recombinant M13 DNA obtained using the traditional calcium chloride method with that obtained by electroporatlon. M13 cloning experiments are often hampered by low yields I.e. insufficient numbers of recombinant plaques are generated. Low concentration of the target DNA, poor ligation due to target DNA impurity and small target fragment size are common factors contributing to this problem. Thus, in cases where such problems cannot be addressed, it is very important to optimise transformation efficiencies. In one such experiment, we attempted to shotgun clone a series of BamH1-Kpn1 fragments from a selected lambda EMBL111 clone containing a 18kb insert of Aeromonas salmonicida chromosomal DNA. The target DNA was digested with BamH1 and Kpn1 and Ugated to BamH1-Kpn1 digested M13mp19 vector. After ligation for 4 hrs at 12°C, the mixture was drop dialysed on a 0.25 urn Millopore filter, and half the mixture was used to transform E.co//TG.1 cells made competent by the calcium chloride method (1). The rest of the ligation mixture was used to transform TG.1 by electroporatlon as follows; Cells were cultured overnight In 2xTY medium (1.6%Bactotryptone,1 % yeast extract, 0.5% NaCI). 10 ml of culture was chilled on ice, harvested at 9,000g, 4°C and washed three times in 10 ml volumes of distilled, deionised sterile H2O. The cell pellet was finally resuspended in approx. an equal volume of H 2 0 and kept on ice. 40 ul of cells were mixed with 5 ul of the ligated DNA (containing1/5 the total ligation mixture) and transferred to a prechPted electroporation cuvette. The cell and DNA mixture was subjected to one pulse at 2.00 k, 25 uF (time constant approx. 4.5msec) using a Gene Puiser ^ m apparatus [ Bio Rad Labs., Richmond, CA ] as described by Dower elal (2). 1 ml SOC buffer (2% Bacto tryptone, 0.5% Bacto yeast extract, 10mM NaCI, 2.5 mM KCI, 10 mM MgC^, 10mM M9SO4,20mM glucose) was immediately added. Dilutions of this mixture were then overiayed on H-agar in seeded top-agar as normal. Table 1 shows the efficiencies obtained with the two methods. In our hands , electroporatlon typically improves M13 transformation efficiencies by between I0 2-io 4 fold. Table 1 Method of Transformation Calcium chloride Electroporation No. of Transformants (pfu) per ng ligated DNA 5 4.8 X10 3 In addition to the considerable increase in the number of transformants obtained by electroporation, this method also …